Molecular diversity and frequency of the diarrheagenic enteric protozoan Giardia duodenalis and Cryptosporidium spp. in a hospital setting in Northern Spain

Abstract

Background: Human giardiosis and cryptosporidiosis are caused by the enteric protozoan parasites Giardia duodenalis and Cryptosporidium spp. Both pathogens are major contributors to the global burden of diarrhoeal disease, affecting primarily children and immunodebilitated individuals in resource-poor settings. Giardiosis and cryptosporidiosis also represent an important, often underestimate, public health threat in developed countries. In Spain only limited information is currently available on the epidemiology of these infections. Molecular data on the diversity, frequency, geographical distribution, and seasonality of G. duodenalis assemblages/sub-assemblages and Cryptosporidium species/sub-genotypes are particularly scarce.

Methods: A longitudinal molecular epidemiological survey was conducted between July 2015 to September 2016 in patients referred to or attended at the Hospital San Pedro (La Rioja, Northern Spain) that tested positive for G. duodenalis (N = 106) or Cryptosporidium spp. (N = 103) by direct microscopy and/or a rapid lateral flow immunochromatographic assay. G. duodenalis infections were subsequently confirmed by real-time PCR and positive isolates assessed by multi-locus sequence genotyping of the glutamate dehydrogenase and β-giardin genes of the parasite. Cryptosporidium species and sub-genotypes were investigated at the 60 kDa glycoprotein or the small subunit ribosomal RNA genes of the parasite. Sociodemographic and clinical parameters of infected patients were also gathered and analysed.

Principal findings: Out of 90 G. duodenalis-positive isolates by real-time PCR a total of 16 isolates were successfully typed. AII (44%, 7/16) was the most prevalent sub-assemblage found, followed by BIV (31%, 5/16) and BIII (19%, 3/16). A discordant genotype result AII/AIII was identified in an additional (6%, 1/16) isolate. No mixed infections A+B were detected. Similarly, a total of 81 Cryptosporidium spp. isolates were successfully typed, revealing the presence of C. hominis (81%, 66/81) and C. parvum (19%, 15/81). Obtained GP60 sequences were assigned to sub-type families Ib (73%, 59/81) within C. hominis, and IIa (7%, 6/81) and IId (2%, 2/81) within C. parvum. A marked inter-annual variation in Cryptosporidium cases was observed.

Conclusions: Human giardiasis and cryptosporidiosis are commonly identified in patients seeking medical care in Northern Spain and represent a more important health concern than initially thought. Assemblage A within G. duodenalis and sub-genotype IbA10G2 within C. hominis were the genetic variants of these parasite species more frequently found circulating in the population under study. Molecular data presented here seem to suggest that G. duodenalis and Cryptosporidium infections arise through anthroponotic rather than zoonotic transmission in this Spanish region.

Fig 1. Temporal and geographical distribution of G. duodenalis and Cryptosporidium spp. infections in La Rioja, Northern Spain (2015–2016).

Panel A: Map of Spain indicating the exact location of the Autonomous region of La Rioja. Panel B: Distribution of G. duodenalis and Cryptosporidium cases according to the primary health care centre of origin and the assemblage/genotype of the parasite species considered. A genotype identification key is provided in the upper right corner of the panel. Panel C: Temporal distribution of G. duodenalis, C. hominis and C. parvum infections through the period of study. An identification key for species and sub-genotypes is provided at the bottom of the panel. Reprinted from Wikimedia Commons, the free media repository.

Fig 2. CONSORT flow diagram showing the flow of information, clinical samples, and diagnostic procedures followed in the present study.

Fig 3. Evolutionary relationships among G. duodenalis sub-assemblages at the GDH locus inferred by a Neighbor-Joining analysis of the nucleotide sequence covering a 383-bp region (positions 88 to 470 of GenBank accession number L40508) of the gene.

The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 iterations) is indicated next to the branches. Bootstrap values lower than 50% were not displayed. The evolutionary distances were computed using the Kimura 2-parameter method. The rate variation among sites was modelled with a gamma distribution (shape parameter = 2). Filled circles represent AII, BIII, and BIV sequences generated in this study. Open circles indicate G. duodenalis sequences of human origin previously reported in other Spanish regions that were included in the analysis for comparative purposes. Spironucleus vortens was used as outgroup taxa.

Fig 4. Evolutionary relationships among C. hominis and C. parvum sub-genotypes at the GP60 locus inferred by a Neighbor-Joining analysis of the nucleotide sequence covering a 810-bp region (positions 47 to 856 of GenBank accession number AY262031) of the gene.

The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 iterations) is indicated next to the branches. Bootstrap values lower than 50% were not displayed. The evolutionary distances were computed using the Kimura 2-parameter method. The rate variation among sites was modelled with a gamma distribution (shape parameter = 2). Filled circles represent Ib, IIa, and IId sequences generated in this study. Open circles indicate Cryptosporidium sequences of human origin previously reported in other Spanish regions that were included in the analysis for comparative purposes. C. meleagridis was used as outgroup taxa.